U.S. patent application number 11/609196 was filed with the patent office on 2008-06-12 for transfection in electronically driven continuous flow.
This patent application is currently assigned to Bio-Rad Laboratories, Inc.. Invention is credited to Charles W. Ragsdale.
Application Number | 20080138876 11/609196 |
Document ID | / |
Family ID | 39498551 |
Filed Date | 2008-06-12 |
United States Patent
Application |
20080138876 |
Kind Code |
A1 |
Ragsdale; Charles W. |
June 12, 2008 |
TRANSFECTION IN ELECTRONICALLY DRIVEN CONTINUOUS FLOW
Abstract
Biological cells and other membranous structures are transfected
in a flow-through system by using a moving charge pattern on a
longitudinal wall of a channel to cause the cells to travel through
the channel due to an electrostatic interaction between the cells
and the moving charge pattern. As the cells travel through the
channel, they pass a transmitter that emits transfection energy
sufficient to make the cell membranes permeable such that exogenous
species in the fluid in which the cells are suspended will enter
the cell interiors.
Inventors: |
Ragsdale; Charles W.;
(Concord, CA) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER, EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Assignee: |
Bio-Rad Laboratories, Inc.
Hercules
CA
|
Family ID: |
39498551 |
Appl. No.: |
11/609196 |
Filed: |
December 11, 2006 |
Current U.S.
Class: |
435/173.1 ;
435/283.1 |
Current CPC
Class: |
C12M 35/02 20130101;
C12N 15/87 20130101 |
Class at
Publication: |
435/173.1 ;
435/283.1 |
International
Class: |
C12N 13/00 20060101
C12N013/00; C12M 1/00 20060101 C12M001/00 |
Claims
1. A method for transfecting a plurality of electrostatically
charged membranous structures with species exogenous to said
structures, said method comprising: (a) introducing a dispersion of
said membranous structures in a liquid solution of said exogenous
species into a channel to which is mounted a transfection energy
transmitter, said channel comprising a longitudinal wall with a
linear array of electrically chargeable surface regions; (b)
electrically charging said surface regions in succession to produce
electrostatic forces between said surface regions so charged and
said membranous structures and to thereby cause said membranous
structures to travel in a direction along said longitudinal wall
and past said transfection energy transmitter; and (c) as each said
membranous structure passes said transfection energy transmitter,
actuating said transfection energy transmitter to achieve said
transfection.
2. The method of claim 1 wherein step (b) comprises imposing a
moving electrostatic charge pattern on said surface regions, said
charge pattern comprising a membranous structure attracting charge
on a first number of surface regions and a membranous structure
repelling charge on a second number of surface regions upstream of
said first number relative to said direction of travel.
3. The method of claim 2 wherein said first number of surface
regions is two or more and said surface regions of said first
number are adjacent.
4. The method of claim 2 wherein said first number of surface
regions is two or more and said charge pattern comprises said
membranous structure attracting charge on two surface regions
separated by an uncharged region.
5. The method of claim 1 wherein said surface regions are
sufficiently small to cause said membranous structures to travel
past said transfection energy transmitter in a single file.
6. The method of claim 1 wherein said membranous structures are
negatively charged biological cells and step (b) comprises imposing
a positive charge to said surface regions in succession.
7. The method of claim 2 wherein said membranous structures are
negatively charged biological cells and said membranous structure
attracting charge is a positive charge and said membranous
structure repelling charge is a negative charge.
8. The method of claim 1 wherein said transfection energy
transmitter is a pair of electroporation electrodes.
9. The method of claim 8 wherein said electroporation electrodes
are a selected pair of said electrically chargeable surface
regions, and step (c) comprises imposing an electroporation
potential between said selected pair.
10. The method of claim 9 wherein said electroporation potential is
achieved by imposing charges on said selected pair of electrodes
that are at least 10 times the charge imposed in step (b).
11. The method of claim 8 wherein said electroporation electrodes
are positioned on opposing sides of said channel.
12. The method of claim 1 wherein said transfection energy
transmitter is a laser diode.
13. The method of claim 1 wherein said transfection energy
transmitter is a combination of a pair of electroporation
electrodes and a laser diode.
14. The method of claim 1 wherein step (b) comprises electrically
charging said surface regions in succession at a rate causing said
membranous structures to travel singly past said transfection
energy transmitter at a rate exceeding 10 structures per
second.
15. The method of claim 1 further comprising detecting locations
and sizes of said structures by measuring resistance to electric
current at said surface regions.
16. The method of claim 1 further comprising detecting locations
and sizes of said structures by interception of light beams through
said channel.
17. The method of claim 1 wherein step (b) comprises electrically
charging said surface regions in succession at a rate causing said
membranous structures to travel singly past said transfection
energy transmitter at a rate of from 100 structures per second to
10,000 structures per second.
18. Apparatus for subjecting a plurality of electrostatically
charged bodies in succession to transfection, said apparatus
comprising: a channel to which is mounted a transfection energy
transmitter, said channel bounded by a longitudinal wall bearing a
linear array of electrically chargeable surface regions;
transfection means for energizing said transfection energy
transmitter to create an energy field sufficient to cause
transfection of said electrostatically charged bodies when said
bodies are within said energy field; and conveying means for
conveying said electrostatically charged bodies in succession
through said energy field by electrically charging said surface
regions in succession to produce a moving charge pattern of
electrostatic forces that attract said electrostatically charged
bodies.
19. The apparatus of claim 18 wherein said transfection energy
transmitter is comprised of electroporation electrodes and said
energy field is an electric field.
20. The apparatus of claim 19 wherein said electroporation
electrodes are a selected pair of said electrically chargeable
surface regions and said transfection means are means for
electrically charging said selected pair of surface regions to
charges that are at least 10 times the charges applied by said
conveying means.
21. The apparatus of claim 19 wherein said transfection energy
transmitter is a laser diode and said energy field is a light
energy field.
22. The apparatus of claim 19 wherein said transfection energy
transmitter is a combination of electroporation electrodes and a
laser diode and said energy field is a combination of an electric
field and a light energy field.
23. The apparatus of claim 18 wherein said electrically chargeable
surface regions are sufficiently small to cause said bodies to
travel through said energy field in a single file.
24. The apparatus of claim 18 wherein longitudinal wall is a
surface of a semiconductor material and said electrically
chargeable surface regions are discrete doped domains in said
semiconductor material.
25. The apparatus of claim 24 wherein said doped domains have
center-to-center spacings of from about 0.1 micron to about 10
microns.
26. The apparatus of claim 24 wherein said doped domains have
center-to-center spacings of from about 0.3 micron to about 3
microns.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] This invention lies in the field of transfection of
membranous structures such as biological cells, liposomes, and
vesicles with species that are exogenous to the structures. In
particular, this invention relates to the mobilization of the
membranous structures to produce a continuous-flow transfection
system.
[0003] 2. Description of the Prior Art
[0004] Transfection is of value to research biologists and
biochemists in the performance of various investigations and
procedures, including siRNA experiments and research using cDNA
libraries, and various other clinical and research procedures. Some
of the most advanced transfection technology is that involving
electroporation, i.e., the application of electric field pulses
through a suspension of the structures in a liquid solution of the
exogenous species to render the membranes of the structures
temporarily porous and thereby allow the species to penetrate the
membrane. As the value of transfection is increasingly recognized
and it use expands, certain concerns have limited its
applicability. One such concern is the efficiency of the procedure,
which is generally low and highly variable due to the tendency of
the membranous structures to aggregate, the different orientations
of the structures and the differences in exposure of the structures
to the electric field with different orientations, and the
shielding effect of individual structures which limits the exposure
of the shielded structures to the field and to the exogenous
species.
[0005] Another concern is throughput, particularly in situations
where the transfection is to be performed on large volumes of cells
or other membranous structures. Certain high-throughput
applications are those involving multitudes of samples where
different cell types or different exogenous species, or both, are
to be subjected to the procedure simultaneously. To accomplish
this, electroporation plates that accommodate large numbers of
samples have been designed. Descriptions of such plates are found
in International Patent Application Publication No. WO 2004/050866
A1, entitled "Large-Scale Electroporation Plates, Systems, and
Methods of Use" (Genetronics, Inc., applicant; Gamelin, A., et al.,
inventors), published under the Patent Cooperation Treaty on Jun.
17, 2004; and in U.S. Provisional Patent Application No.
60/771,994, filed Feb. 10, 2006, entitled "Apparatus for
High-Throughput Electroporation" (inventors Ragsdale, C. W., et
al.) and commonly owned herewith. Other high-throughput
applications are those that simply involve a large volume of
membranous structures, more for example than can be accommodated in
a single electroporation cuvette.
[0006] In the majority of the literature on electroporation and
transfection in general, and all of the commercially available
electroporation systems, the procedure is performed in cuvettes in
a batchwise format. With the high degree of manipulation and
repetition involved in batchwise procedures, together with the size
limitations of the typical cuvette, the processing of large volumes
of sample and large numbers of structures is costly and prone to
error. Continuous use of an electroporation chamber designed for
batchwise use entails a risk of overheating of the chamber and
irreparable rupture of the membranes. Continuous-flow systems have
been contemplated but with only limited application. Descriptions
of continuous flow systems appear in Nicolau et al. (CBR
Laboratories, Inc.), U.S. Pat. No. 5,612,207, "Method and Apparatus
for Encapsulation of Biologically-Active Substances in Cells,"
issue date Mar. 18, 1997, and Meserol, P. (EntreMed, Inc.), U.S.
Pat. No. 6,090,617, "Flow Electroporation Chamber With Electrodes
Having a Crystalline Metal Nitride Coating," issue date Jul. 18,
2000. The electrodes in these patents are elongated strip
electrodes, and electroporation is achieved by pumping the cells
through the space between the electrodes, using a simple mechanical
pump. The electroporation rate is limited by the pump rate, and
there is little or nor control over such factors as the density of
the suspension at any particular point in the flow path and
differences in the exposure of individual cells to the electric
field. A description of another moving system is found in Acker, J.
L., et al., United States Patent Application Publication No. US
2004/0029240 A1, publication date Feb. 12, 2004. The system used b
Acker et al. involves moving electrodes and is not a flow-through
system. The purpose of the moving electrodes is to impose a shear
stress on the cells to cause them to continuously change their
orientation.
[0007] Of further potential relevance to the background of the
present invention is the use of electromagnetic radiation, such as
pulses of light, to achieve transfection. In a manner analogous to
electroporation, exposure of a membranous structure to a pulse of
light energy can result in a transient permeabilization of the
membrane without rupoture of the membrane. As in electroporation,
this is done while the cell is suspended in a solution of a
molecule that is exogenous to the cell, thereby allowing the
molecule to enter the cell through the permeated membrane. A
description of this technique is found in Koller, M. R., et al.
(Oncosis LLC), U.S. Pat. No. 6,753,161 B2, "Optoinjection Methods,"
issue date Jun. 22, 2006. The transient permeabilization effect is
performed while the cells are "substantially stationary."
SUMMARY OF THE INVENTION
[0008] The present invention resides in a system and method for the
transfection of electrically charged membranous structures in a
continuous-flow format by utilizing the electrical charge on the
structures to convey the structures through a channel and past a
transmitter of transfection energy in the channel. By virtue of
their electrical charge, the structures are attracted to opposing
electrical charges on a longitudinal wall within the channel, the
opposing electrical charges being imposed on chargeable surface
regions that are arranged in a linear array and charged in
succession to create a moving charge pattern along the wall. The
moving charge pattern allows the travel of the membranous
structures to be controlled to such an extent that the membranous
structures can be made to establish moving contact with, or very
close proximity to, the wall and to travel in a single file past
the transfection energy transmitter where they will undergo
transfection either one at a time or in groups of preselected size
at preselected time and spatial intervals. As in conventional
transfection, the structures are suspended in a solution of the
exogenous species, and the moving charge pattern also allows each
structure to be exposed to the same electric field without
aggregation of the structures or shielding of one structure by
another. Transfection that is substantially uniform among all of
the structures at a high rate of efficiency is thus achieved, with
sufficient control over the transfection conditions that
destruction of the structures due to excessive energy from the
transmitter is minimal, if not eliminated entirely. Automated
electronic control over the charging of the chargeable surface
regions on the wall also allows the system to accommodate
membranous structures of different sizes and dimensions by
selecting the number and spacing of the regions to be charged in
the moving pattern, to vary the spacing between adjacent membranous
structures, and to vary the number of structures that are exposed
to the transmitter at any point in time, i.e., whether transfection
be performed on only one structure at a time or more than one.
[0009] These and other operations, functions, and advantages of
this invention are explained in further detail below.
BRIEF DESCRIPTION OF THE DRAWING
[0010] The FIGURE is a perspective view of a transfection channel
in accordance with the present invention with a portion of the
channel wall removed to show the interior.
DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS
[0011] The membranous structures to which the present invention is
applicable are bodies that are at least of macromolecular
dimensions and include an enclosing membrane that is impenetrable
to the species of interest under normal conditions. Examples of
such membranous structures are liposomes, vesicles, organelles, and
biological cells. Biological cells include both prokaryotic and
eukaryotic cells, and can be animal cells, plant cells, yeast
cells, human cells, bacteria, or any other similar structures. The
electrostatic charges on the membranous structures can be either
naturally occurring or added by surface functionalization or
complexation. Many biological cells, for example, bear a negative
surface charge in their natural form.
[0012] The term "exogenous species" is used herein to denote any
molecule or cluster of molecules that is not native to or otherwise
present in the membranous structures, or is present inside the
structure but in a limited quantity or at a limited concentration
and whose quantity or concentration within the structure is to be
increased by transfection in accordance with this invention.
Examples of classes of exogenous species are nucleic acids,
polypeptides, carbohydrates, lipids, and small molecules in
general. Examples of nucleic acids are RNA, expression plasmids,
expression cassettes and other expressible DNA. Examples of
polypeptides are antibodies, antibody fragments, enzymes, and
proteins in general. Examples of carbohydrates are non-naturally
occurring metabolites such as isotopically labeled sugars, and
polysaccharides such as labeled dextrans. Liposomes may serve as
exogenous species when the membranous structures are bodies larger
than liposomes. Examples of small molecules are drugs, dyes, and
ligands for endogenous receptors.
[0013] The term "transfection energy" is used herein to denote any
form of energy applied to a membranous structure that will render
the membrane reversibly porous or otherwise permeable for a limited
period of time sufficient to allow exogenous species in the
suspending liquid to penetrate the membrane and enter the interior
of the structure, and to do so without irreparably rupturing the
membrane or otherwise causing permanent damage to the structure.
Examples of transfection energy are electrical energy (resulting in
electroporation), light energy (both from a laser and from
non-laser sources), thermal energy, RF energy, ultrasound, and
electron beam energy. Preferred forms of transfection energy are
electrical energy and laser light energy, applied either
individually or in combination. Electrical energy (electroporation)
is particularly preferred. The "transfection energy transmitter" is
any device or component that will create a field of transfection
energy, preferably one that is focused within a spatial volume of
dimensions that are limited to achieve transfection in a
preselected number of membranous structures. The field can be small
enough to accommodate only one structure at a time, or broad enough
to accommodate a limited plurality such as two or more structures,
or it can be a ray of energy sufficiently narrow to strike only one
structure. Transmitters that are known in the art for each
particular type of energy can be used. For electroporation, the
transmitters can be electrodes; for light or thermal energy, the
transmitters can be laser diodes. Other transmitters for these and
other forms of transfection energy will be apparent to those
skilled in the art.
[0014] The electrically chargeable surface regions on the
longitudinal wall of the channel are fixed, stationary regions that
can be individually and selectively charged, i.e., rapidly switched
between charged and electrically neutral, or between positively
charged, negatively charged, and neutral, by switching means
external to the wall or to the channel. Each region occupies a
fixed position on the wall and can be formed by attachment of an
electrode to the surface of the wall, incorporation of an electrode
material in the construction of the wall, or doping the wall with
ionic species as in conventional semiconductor fabrication
technology. In certain embodiments, the wall is formed of silicon
or other semiconductor material and the regions are strip areas of
the wall doped with chargeable ions.
[0015] The moving charge pattern on the wall preferably consists of
two or more regions bearing charges that attract the membranous
structure, and more preferably two or more such regions in addition
to one or more regions bearing charges that repel rather than
attract the membranous structure. The region(s) bearing the
repelling charge will be positioned upstream (i.e., at the trailing
end) of the region(s) bearing the attracting charge to help propel
the membranous structure forward in the direction of travel through
the channel, the two charges thereby imposing both a pushing force
and a pulling force in combination on each passing structure. The
use of two regions of attracting charge in the charge pattern
serves to provide optimal control of the positions of the
membranous structures at all points during their travel through the
channel, stabilizing the structure across the charged regions when
the spacing between the charged regions is approximately equal to
or slightly smaller than the length or diameter of the membranous
structure. In certain embodiments, the system is adaptable by
allowing the operator to select among different charge patterns to
accommodate membranous structures of different sizes. Two regions
bearing attractive charges can thus be separated by one or more
uncharged regions in the charge pattern, the number of intervening
uncharged regions determining the spacing of the charged regions.
In most cases, best results will be obtained with a
center-to-center spacing of from about 0.1 micron to about 10
microns, and preferably from about 0.3 micron to about 3 microns,
between regions bearing the attractive charge.
[0016] The same surface regions that move the membranous structures
can also be used to transfect. This is explained in detail below.
The surface regions can also serve as detectors of the sizes of the
membranous structures. As a structure moves across adjacent surface
regions, current can be passed through the regions, and the
resistance to the current measured. The resistance when a cell or
other membranous structure is touching a given surface region will
differ from the resistance when no structure is touching the
region. The number of adjacent regions that the structure is in
contact with at a given point in time thus indicates the size of
the structure. Size can also be detected by optical sensors, such
as for example LEDs (light-emitting diodes) in conjunction with
phototransistors positioned to receive the beams of light from the
LEDs through the moving path of the membranous structures.
Regardless of the mechanism, once the sizes of individual
membranous structures are determined, the charge pattern can be
adjusted to accommodate the structure size and thereby provide
optimal control over the movement of the structures.
[0017] As noted above, the chargeable surface regions can be formed
by integrated circuit techniques such as doping of metallization
etched into a semiconductor surface. The electronic drivers that
govern the charge pattern and its movement can be such commonly
known components as transistors, IGBTs (insulated gate bipolar
transistors) and power FETs (field effect transistors). As an
example of a charging protocol to create a moving charge pattern, a
first series of surface regions, for example, the four such regions
at the entry to the channel, can be made positive to attract a
biological cell, which bears a natural negative charge. When a cell
is sensed by electrical or optical means as described in the
preceding paragraph, or after a specified period of time has
passed, the first region is turned off (switch to electrical
neutrality) and then given a negative charge as the fifth region
(previously neutral) is given a positive charge. This continues in
succession down the array of surface regions.
[0018] The term "linear array" is used herein to indicate
electrically chargeable regions that are arranged in a line, which
can be either curved or straight, such that when the charge pattern
is moved along them they direct the membranous structures along a
unidirectional path of travel. In most cases, a straight-line array
will be most convenient. Two or more parallel linear arrays can be
present, doubling or otherwise multiplying the capacity of the
channel and the rate of transfection.
[0019] The system can be designed to accommodate either a single
structure at a time passing through the channel or multiple
structures. When the channel is long enough to accommodate two or
more structures, the charge protocol will include a number of
moving charge patterns equal to the number of structures. The
spacing between adjacent charge patterns will preferably be
sufficient to avoid interference between successive structures in
their movement through the channel and in their exposure to the
transfection energy from the transmitter. A spacing equal to ten or
more structure diameters, and preferably fifty or more, will
provide the best results in most cases.
[0020] The charge pattern can be designed to cause the membranous
structures to travel in a single file, double file, or more. Travel
in a single file is generally sufficient in most applications and
can be achieved by limiting the dimensions of the charged regions,
the dimensions of the channel, or both. The travel velocity and
number of structures passing through the channel per unit time can
also vary. Preferably, the rate of travel is high enough to cause
ten or more structures per second to pass the transfection energy
transmitter, preferably 100 to 10,000 structures per second, and
most preferably 300 to 3,000 structures per second.
[0021] The dimensions of the channel will nevertheless be large
enough to allow the structures to flow freely through the channel
without clogging the channel. A channel width or diameter of at
least about 10 microns, preferably about 20 microns or greater,
will be suitable in most cases, particularly for biological
cells.
[0022] The transfection energy transmitter is positioned at a fixed
location in the channel so that membranous structures during the
course of their travel through the channel will come within the
range of the transmitter. When the transmitter is a pair of
electrodes to cause transfection by electroporation, the electrodes
can be a dedicated pair of electrodes, either on the same side of
the channel or on opposing sides. For electrodes on the same side
of the channel, two of the chargeable surface regions, either
adjacent or in close proximity, can also serve as the
electroporation electrodes by imposing a higher voltage between
them for electroporation. For example, when simply causing the
travel of a structure, the two surface regions can be charged at
the same polarity with a charge in the millivolt range, and when
the structure is in position for electroporation, the two regions
can be charged at opposite polarities with charges in the volt
range. In view of the very small dimensions of the system and the
close proximity of the membranous structures to the electrodes, a
typical voltage range for electroporation will be within the range
of 0.3-30 V, preferably 1-5 volts, and this will typically be 10 to
1,000 times the voltage for travel. When electroporation electrodes
are used that are distinct from the moving charge pattern surface
regions, the former can be placed between an adjacent pair of the
latter. When laser diodes or other transmitters that produce
temperature- or light-induced poration are used, they can likewise
be placed on one side of the channel or on opposing sides, and most
effectively between an adjacent pair of chargeable surface regions.
Laser diodes will require little or no optics in view of their
close proximity to the membranous structures.
[0023] While the features defining this invention are capable of
implementation in a variety of constructions, the invention as a
whole will be best understood by a detailed examination of a
specific embodiment. One such embodiment is shown in the attached
FIGURE.
[0024] The FIGURE depicts a continuous-flow transfection apparatus
11 which includes a channel 12 shown with parts of the wall removed
to make the interior of the channel visible. The channel is open at
both ends, with one end designated an entry end 13 and the other an
exit end 14. The inner surface 15 of one longitudinal wall contains
a series of regularly spaced regions 16 that serve as the
chargeable regions, with adjacent regions spaced apart from each
other. Transfection energy transmitters 17, 18 are positioned on
opposing sides of the channel, one 17 on the same wall as the
chargeable regions and between two adjacent chargeable regions, and
the other 18 directly opposite on the opposing wall. Three
membranous structures (negatively charged biological cells) 21, 22,
23 are shown moving through the channel in the direction indicated
by the arrows. The moving charge pattern in this case consists of
three adjacent surface regions, the first located closest to the
entry end 13 and bearing a negative charge that repells the cells,
and the second and third bearing a positive charge that attracts
the cells. To draw a cell into the channel, the region 24 nearest
the entry end is positively charged. As the second and third
regions become positively charged, the region 24 nearest the entry
end is given a negative charge to urge the cell further into the
channel. The charge pattern then travels through the channel,
drawing the cell with it, past the transmitters 17, 18.
[0025] While the foregoing description describes various
alternatives to the components shown in the FIGURES, still further
alternatives will be apparent to those who are skilled in the art
and are within the scope of the invention.
[0026] In the claims below, the terms "a" and "an" are intended to
mean "one or more." The term "comprise" and variations thereof such
as "comprises" and "comprising," when preceding the recitation of a
step or an element, are intended to mean that the addition of
further steps or elements is optional and not excluded. All
patents, patent applications, and other published reference
materials cited in this specification are hereby incorporated
herein by reference in their entirety. Any discrepancy between any
reference material cited herein and an explicit teaching of this
specification is intended to be resolved in favor of the teaching
in this specification. This includes any discrepancy between an
art-understood definition of a word or phrase and a definition
explicitly provided in this specification of the same word or
phrase.
* * * * *